Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity. A mechanistic understanding of the cleavage of intact collagens has been pursued for many years; the results of such studies could lead to the development of truly selective MMP inhibitors. Our laboratory developed triple-helical peptides (THPs) as MMP substrates, with the goal of using these models to dissect collagenolytic behavior and develop selective MMP inhibitors. In the most recent funding period of the present project (04/01/08-present), we have experimentally derived the initial steps of collagenolysis and identified specific residues involved in this process, quantified the roles of specific collagen residues in MMP substrate specificity, and developed selective MMP inhibitors based on secondary binding sites (exosites), and demonstrated the in vivo use of such inhibitors. Our studies also revealed intriguing, unexpected behaviors of collagenolytic MMPs, such as MMPs bind the triple-helix using slightly different orientations, one can use exosite binding THPs to inhibit some, but not all, proteolytic activities for a given enzyme, which could significantly reduce side effects, and substrate selectivity seen for single-stranded peptides is not observed in the triple-helix. The research plan described herein focuses on further development of triple-helical probes for teasing out collagenolytic MMP sequence specificities, identifying selective MMP inhibitors, and advancing the mechanism of collagenolysis. To achieve these goals we propose to (a) identify THP sequence preferences for the MMP family utilizing positional scanning combinatorial libraries, (b) design and characterize MMP selective, homotrimeric and heterotrimeric triple-helical transition-state analog inhibitors, and (c) explore the latter steps of collagenolysis usin state-of-the-art NMR spectroscopic techniques and design exosite binding THPs. Select inhibitors will be tested in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancers such as melanoma and breast carcinoma (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP-8).